Fungal pathogens present a severe risk to food systems; however, complex crop–microbe interactions are challenging to study using tools developed for model species. In particular, efficient screening and rapid assessment of microbial effectors is hindered by a lack of cloned resistance (R) genes and difficulty in validating large numbers of predicted effector candidates. This chapter describes a protocol for preparing wheat protoplasts to enable positive identification of host defense induction without overexpression of a cloned R gene, increasing the available pool of host resistance genes for screening. The assay uses polyethylene glycol (PEG)-calcium-mediated transient transfection to introduce candidate effector gene constructs into wheat protoplasts, with a defense-activated reporter for inducing a positive readout with internal normalization, indicating host recognition. This protocol provides a valuable tool for the study of host–pathogen interactions in wheat, contributing to improved resources for the development of disease-resistant crops and genome-informed pathogen surveillance.